Endothelial cells play an important role in determining the risk for atherosclerosis and coronary artery disease (CAD). Exercise may restore/preserve a healthy endothelium and limit atherosclerosis. An important part of this project will be to determine in a well-controlled porcine model, the effects of exercise on the expression of a group of proteins known to have a central role in determining the risk of vascular disease. The experiments are focused on a set of putative PPAR-responsive mRNAs/proteins and lipoprotein lipase (LPL). LPL is a rate-limiting enzyme for the metabolism of triglyceride-rich lipoproteins, and as such, high levels of LPL activity have been associated with the metabolic demands of exercise. The effect of LPL-dependent lipolysis on the phenotype of endothelial cells is still largely unclear and needs to be elucidated. A strength of these experiments is that they employ a multifaceted and collaborative approach to accomplish a definitive set of hypothesis-driven studies. Molecular studies will determine the mRNA and protein targets regulated directly by LPL-dependent signaling in endothelial cells (Aim 1). This aim will use a wide-range of experimental culture conditions involving different amounts of LPL activity, purified lipoprotein substrates, RNA interference for signaling studies, and pharmacological ligands to compare with lipoprotein derived ligands. Aim 1 will also test for interactions during exposure to TNFa as a pro-inflammatory cytokine. More physiological studies will be performed in isolated conduit arteries while determining the interactions between chemical signals caused by experimentally reducing and increasing LPL activity, and the hemodynamic signaling caused by altering flow rate over a wide range (Aim 2). Exercise training studies will determine how LPL and a group of related proteins are regulated in different vascular sites (Aim 3). Aim 3 will be a very complete examination of exercise responses because it will determine the effects of training in pigs fed a normal fat diet before there is a risk of CAD, in a model of risk for early CAD, and in more advanced CAD. These studies will provide important novel insights about regulation of LPL activity, and the mechanisms involved in determining a healthy endothelial cell phenotype.